Pharmacokinetic characteristics of mesenchymal stem cells in translational challenges

Over the past two decades, mesenchymal stem/stromal cell (MSC) therapy has made substantial strides, transitioning from experimental clinical applications to commercial products. MSC therapies hold considerable promise for treating refractory and critical conditions such as acute graft-versus-host disease, amyotrophic lateral sclerosis, and acute respiratory distress syndrome. Despite recent successes in clinical and commercial applications, MSC therapy still faces challenges when used as a commercial product. Current detection methods have limitations, leaving the dynamic biodistribution, persistence in injured tissues, and ultimate fate of MSCs in patients unclear. Clarifying the relationship between the pharmacokinetic characteristics of MSCs and their therapeutic effects is crucial for patient stratification and the formulation of precise therapeutic regimens. Moreover, the development of advanced imaging and tracking technologies is essential to address these clinical challenges. This review provides a comprehensive analysis of the kinetic properties, key regulatory molecules, different fates, and detection methods relevant to MSCs and discusses concerns in evaluating MSC druggability from the perspective of integrating pharmacokinetics and efficacy. A better understanding of these challenges could improve MSC clinical efficacy and speed up the introduction of MSC therapy products to the market.


INTRODUCTION
Mesenchymal stem/stromal cell (MSC), which can be harvested and expanded from various adult and perinatal tissues, such as adipose tissue, bone marrow (BM), dental pulp, and umbilical cord (UC), 1 exhibits diverse pharmacological effects, including immunomodulation, angiogenesis, and regenerative properties, rendering them promising for therapeutic applications. 2MSC therapy, which exerts the therapeutic potential of living cell preparations, has experienced explosive growth in both clinical deployment and expansion within the pharmaceutical marketplace for decades. 3,4everal MSC therapies approved by regulatory agencies and introduced to the commercial market have attracted increasing public attention.These include the successful approval of Prochymal (Remestemcel-L) in 2012 for treating graft-versus-host disease (GvHD) in Canada and Alofisel in 2018 for the treatment of Crohn's disease in Europe (Table 1).The significant clinical benefits of MSC therapies are inspiring for further exploration and development.
Many previous studies disconnected basic biomedical research and the potential druggability of MSCs.A limited understanding of the dynamic biodistribution and fate of MSCs within the body threatens the development of MSC therapies.The evaluation of the safety and efficacy of MSC therapy is limited by its unfavorable pharmacokinetic (PK) and pharmacodynamic (PD) properties. 5harmacokinetics, which describes how the body interacts with a drug, involves the processes of absorption, distribution, metabolism, and excretion, detailing the drug's movement into, through, and out of the body over time. 6Unlike conventional drugs, MSCs can naturally migrate, localize, and even proliferate in specific tissues or compartments. 7In this review, we can consider the biodistribution of MSCs within the body as "distribution" and their fate as "metabolism and excretion".Furthermore, MSCs are typically distinct from other cell therapies because their therapeutic efficacy depends not only on cell-to-cell contact but also on what is often referred to as a "hit-and-run" (or "touch and go") mechanism-that is, through their rapid migration to damaged tissues and subsequent clearance following the release of paracrine effectors from their secretome, including soluble cytokines, growth factors, hormones, and miRNAs 8,9 or the transfer of mitochondria to target cells through tunneling nanotubes, 10,11 potentially leading to long-lasting effects. 12Thus, the lack of control over the localization, migration, and fate of MSCs is a translational challenge for MSC therapies. 13umerous studies have shown that secretome-derived bioproducts containing bioactive molecules (such as proteins, lipids, and nucleic acids) and MSC-derived extracellular vesicles (EVs) retain the biological activity of parent MSCs and demonstrate similar therapeutic effects. 146][17] In this review, we first outline the key molecules involved in the motility of MSCs.We then provide a brief introduction to the various methods for tracking MSCs in vivo, highlighting their advantages and limitations.The core of the article focuses on the biodistribution and fate of MSCs in vivo, assessing the relationship between the kinetic characteristics and druggability of MSCs.Finally, we discuss the prospects of future opportunities for the clinical and commercial use of MSCs.

THE LANDSCAPE IN CLINICAL MSC THERAPIES
The success of clinical trials depends on understanding the mechanisms of drugs, but the clinical application of MSCs has frequently surpassed the understanding of their underlying mechanisms.A search using the keyword "mesenchymal stem/ stromal cell" on ClinicalTrials.govrevealed more than 1600 related clinical trials, with more than 1500 clinical trials employing MSCs as a therapeutic intervention.With over 500 clinical trials of MSCs expected to be conducted by 2023, there is a wealth of information that can be leveraged to enhance our understanding of the factors influencing their successful implementation in human subjects (Table 2).These clinical trials are mostly in phase I, phase II, or combined phase I/II trials.Only a small percentage of trials are in phase III (comparing newer treatment approaches with standard or most well-known treatments) or phase II/III.Overall, MSCs appear to be well tolerated, with the majority of trials reporting no adverse reactions in the midterm.Only a few trials indicated mild and transient adverse reactions during the injection period.While MSCs exhibit a favorable safety profile, they often struggle to demonstrate significant efficacy in humans. 18Notably, no MSC therapies have received approval from the National Medical Products Administration (NMPA) of China or the U.S. Food and Drug Administration.
Representative registered clinical trials of MSC therapies Many completed clinical trials have demonstrated the efficacy of MSC infusion in treating a variety of diseases, such as GvHD, multiple sclerosis (MS), Crohn's disease (CD), amyotrophic lateral sclerosis (ALS), myocardial infarction (MI), and acute respiratory distress syndrome (ARDS), among others (Table 2).Kabat et al. classified the indications for clinical trials into 14 groups, with those that could not be classified or had too few cases classified as "Other". 19Among these clinical trials, MSC therapies for "neurological" diseases (17%) and "joint" diseases (15%) are the most common in registered indications.Cardiovascular disease (8.8%) and GvHD (8.3%) are closely related to this disease, with a higher percentage of phase III clinical trials.Only 84% explicitly specify the route of administration in MSC clinical trials.
Clinical comparison of MSC therapy and cell-free therapy Compared to MSC therapy, cell-free therapy utilizing the MSC secretome involves the delivery of multiple bioactive molecules rather than intact cells. 20Assessing the safety and efficacy of MSCconditioned medium will be considerably less complicated. 21urthermore, the production of conditioned medium is more economical, as it can be scaled up for mass production utilizing existing MSC populations under current good manufacturing practice conditions.Nonetheless, cell-free therapy products share similarities with conventional drugs in that they may necessitate prolonged administration and could result in the development of drug resistance and adverse reactions. 22However, cell therapy offers the distinct advantage of potentially conferring enduring effects with a single treatment that can last for several months, reducing the need for frequent medication and enhancing the resilience of the treatment regimen. 23Additionally, to enhance the efficacy of MSC therapy and their homing ability, the singular or combined application of bioengineering techniques shows promising potential to significantly overcome the current clinical challenges.

Impact of different administration routes
The pharmacokinetics and biological properties of infused MSCs may be influenced by the route of administration. 13,24The administration routes in MSC clinical trials mainly include intravenous, intracardiac, intra-articular, intramuscular, intraosseous, intrathecal, intra-arterial, and implantation.Among the eight most frequently employed routes, intravenous infusion accounts for 43%. 19This preference for intravenous infusion is attributed to its ease of administration, low invasiveness, and high repeatability, making it the most frequently utilized method in treatment. 25ocal administration is commonly employed because it allows direct delivery to the disease site.Forty-nine percent of registered MSC clinical trials utilized local delivery by 2018. 26Many MSC therapies are locally administered for various diseases, including lumbar pain, anal fistula, and chronic heart failure, in late-stage clinical trials. 27Local administration of MSCs is a more controllable approach, making it easier to access disease sites and often yielding better therapeutic responses. 28However, in certain cases, less than 5% of the administered cells have been observed to remain at the injection site just hours after transplantation. 29oreover, local administration of MSCs also results in secondary distribution but is limited to locally vascularized areas such as the heart. 30The poor persistence of MSCs after local administration is due to several factors, such as cell death from the inhospitable microenvironment at the disease site and ineffective integration into the tissue. 31Compared to systemic administration, direct injection of MSCs into damaged tissue sites lacks systemic homing processes, but the persistence of MSCs in target tissues remains similar to that of systemic administration.
Systemic administration of MSCs is more minimally invasive and can avoid tissue calcification issues compared with local injection. 32Among the approved MSC products, treatments for GvHD and spinal cord injury are administered systemically.Although many studies in animals and humans have shown that intravenously infused MSCs tend to stick to the lungs, 33 systemic Table 1.MSC products approved in different countries administration of MSC therapy has been used in most clinical trials by 2023.Due to nonspecific uptake by blood vessels in nontarget tissues, higher doses are needed for MSCs to achieve effective treatment levels in target organs and tissues.The administration of 10 5 -10 7 MSCs per kilogram is required based on patient weight, 34,35 leading to widespread biodistribution in tissues and organs such as the lung, liver, and gastrointestinal tract. 36An increasing number of MSC clinical trials are using systemic administration, highlighting the need for a comprehensive understanding of the pharmacokinetics of MSCs in the body.The detailed process of systemic administration of MSCs in vivo can be found in the next section.

MOLECULES OF GOVERNING MSC MOTILE POTENCY DURING HOMING
After systemic administration, MSCs enter the blood circulation and cross the vascular endothelium at lesion sites through four steps: (1) tethering and rolling, (2) activation, (3) adhesion, and ( 4) transmigration (Fig. 1).This process involves various proteins that regulate MSC motility and interactions with endothelial cells (ECs).The ligands expressed on the surface of MSCs mediate their rolling action on the vascular wall upon binding to selectins.The G protein-coupled chemokine receptors on MSCs receive activation signals, leading to integrin-dependent adhesion to the endothelium.Finally, MSCs secrete proteases to break down the EC barrier, completing the homing process. 7,37,38lectin ligands The homing process is initiated through tethering events mediated by selectin ligands on MSCs and selectins on ECs (Table 3), with MSCs rolling after attaching to ECs. 39 Hematopoietic cell E-selectin/L-selectin ligand (HCELL), a functional form of CD44 containing an sLex-like epitope, binds to E-selectin/L-selectin and functions as a marker of MSC motility. 40,41P-selectin also plays a role in mediating the rolling of MSCs through interacting with P-selectin glycoprotein ligand (PSGL-1), 42 and blocking P-selectin decreases the binding of MSCs to ECs. 39 Suila et al. reported that Galectin-1, rather than PSGL-1, tends to serve as a marker of MSC motility, as MSCs preferentially interact with P-selectin on ECs through Galectin-1, which is independent of the sLex epitope. 43The complex interplay of various selectin ligands is crucial for the efficient adhesion of MSCs to ECs.

Integrins
Integrins are transmembrane receptors that act as interfaces between extracellular and intracellular compartments and facilitate cell-extracellular matrix (ECM) adhesion. 44MSCs express a broad spectrum of integrins, including β1, β2, β3, β5, α1, α2, α3, α4, α5, α6, α7, α8, α9, αX, αV, and αD 45 (Table 3).The integrin VLA-4 (α4β1), which is composed of CD49d (α4) and CD29 (β1), is highly expressed on human MSCs 46 and plays a crucial role in the adhesion and rolling of MSCs.Vascular cell adhesion molecule-1 (VCAM-1) is an immobilized protein expressed on ECs that can bind to CD29, resulting in fast-rolling interactions under appropriate shear stress. 47Blocking CD29 or VCAM-1 abolishes MSC rolling, indicating that these rolling interactions depend on adhesion.This subsequently diminishes MSC migration to the injured liver and ischemic myocardium. 40Compared to that of CD29, the amount of CD49d on MSCs is insufficient, which may limit the homing ability of MSCs. 48Overexpression of CD49d enhances transendothelial migration in vitro and increases homing to the BM in vivo, 49 demonstrating that VLA-4 is a suitable marker for MSC motility.Notably, MSCs also express intercellular cell adhesion molecule (ICAM-1, the ligand for αLβ2) naturally, but the significance of ICAM-1 in MSC homing still needs to be clarified.Integrin, a significant molecule involved in MSC

Chemokine receptor family
Chemokines and their receptors are now recognized as important mediators of MSC homing.Human chemokines are a superfamily of 48 ligands that bind to 19 different G protein-coupled chemokine receptors, regulating cell proliferation, differentiation, chemotaxis, and other physiological processes. 50They can be categorized into several groups depending on their functions. 51,52nflammatory chemokines are upregulated during inflammation, promoting leukocyte recruitment and migration toward injury or infection sites. 53Homeostatic chemokines regulate adaptive immune responses and contribute to stem cell systemic migration. 51MSC activation is triggered by G protein-coupled chemokine receptors that respond to inflammatory signals, with chemokines binding to chemokine receptors on MSCs, leading to integrin recruitment and increased adhesiveness. 54In addition, the increased integrin affinity is attributed to rapid lateral mobility triggered by chemokines. 55MSCs express a broad panel of chemokine receptors, including CCR1-7, CCR9-10, CXCR1-6, and CX3CR1, [56][57][58][59][60] but the expression of most these receptors is low and decreases after in vitro expansion, which limits studies on MSC homing and therapeutic efficacy. 61The chemokinechemokine receptor network also plays significant roles in the tropism of MSCs from the bloodstream toward different organs or tissues in response to inflammation or injury (Table 3).
CCR1.CCR1 on MSCs is a key marker of motile potency, with a broad spectrum of chemokines acting as natural ligands.CCL3, which is produced by lymphocytes, monocytes, and macrophages in response to proinflammatory agents/cytokines, can accelerate MSC migration by interacting with CCR1 on MSCs. 62Stimulating CCR1-overexpressing MSCs with CCL5 increased the ability of MSCs to home to injured sites and restored cardiac function. 63CL7, which is significantly expressed in the urethras of rats during stimulated birth injury, is a ligand for CCR1, and pretreatment combined with CCR1-MSC overexpression increases MSC engraftment to the mid-urethra. 64The lesioned hippocampus expresses increased CCL2, which recruits human olfactory ectomesenchymal stem cells (OE-MSCs) in vitro. 65CCL2 may bind to and activate CCR1 and CCR10 in OE-MSCs through an unknown migration pathway.CCR2.Previous studies have shown that CCL2 (the ligand of CCR2) is widely present in certain inflammatory diseases and monocyte recruitment in injured tissue mainly depends on CCR2. 66Therefore, CCR2 on MSCs may also play an important role in migration.8][69][70][71] Huang et al. showed that CCR2 overexpression on MSCs enhanced their homing to the ischemic hemisphere and improved therapeutic outcomes. 69Moreover, CCR2 also plays a crucial role in regulating the motility of MSCs and promoting their recruitment to liver injury sites. 72CCL7, another ligand for CCR2, could induce MSCs to migrate toward the injured myocardium, resulting in beneficial remodeling in the infarct zone. 71R3 and CCR4.MSCs typically do not express CCR3, but following infection with Kaposi's sarcoma-associated herpesvirus (KSHV), they exhibit increased CCR3 expression and enhanced migration ability in vitro.Knockdown of CCR3 or blockade of its ligand CCL5 can partially suppress MSC migration, suggesting that the CCL5-CCR3 axis may promote MSC homing. 73Treatment with the cationic molecule polyethylenimine (PEI) dose-dependently increased CCR4 expression on MSCs, and the brain homing efficiency of MSCs significantly increased after intravenous administration of PEI in a rat model of brain injury. 74R6 and CCR7.Overexpressing CCR6 enhances MSC migration to skin wound sites. 75BM-MSCs expressing CCR6 can transmigrate across ECs in response to CCL20. 76Transfected MSCs with enhanced CCR7 expression showed improved migration to secondary lymphoid organs in response to SLC/CCL21, leading to prolonged survival in GvHD mice. 77Additionally, injection of CCL21 into the periphery of wounded skin significantly increased MSC recruitment to wound sites and accelerated wound closure in skin wound models. 78R9 and CCR10.CCL25 acts as a chemoattractant for various immune cells and is a ligand for CCR9. 79CCL25 has been found to mediate the migration of MSCs expressing different levels of CCR9 in vitro. 80In addition, CCL27 (the ligand of CCR10), a skin-specific chemokine, is predominantly expressed by keratinocytes in the epidermis of skin (injured/uninjured/exudates), and its upregulation recruits MSCs to migrate toward wounded skin. 81In vivo studies have shown that systemically administered MSCs overexpressing CCR10 migrate to the skin in response to CCL27. 82CR1 and CXCR2.CXCL8 (also known as interleukin 8, IL-8) is an inflammatory chemokine that can interact with CXCR1 and CXCR2. 83Ringe et al. found that MSCs migrate to zones with high concentrations of CXCL8 in a dose-dependent manner through in vitro chemotaxis assays. 83This process was observed in gliomas, in which CXCL8 was identified as an inducer of human umbilical cord blood-derived MSC migration toward glioma cells both in vitro and in vivo. 84,85XCR2, a molecule with various ligands, is crucial for leukocyte migration toward inflamed BM sites. 86Genetic modification can upregulate CXCR2 expression on MSCs, enhancing their mobilization and accelerating healing. 87,88CXCL7 recruits human BM-MSCs in vitro and may influence their migration through the IL-8 receptor signaling pathway.The activation of chemokines that stimulate CXCR1 and CXCR2 suggests a synergistic mechanism for CXCL7-driven MSC migration. 89CR4.CXCL12 (SDF-1)-CXCR4 has already been the most widely studied MSC chemotactic axis. 90CXCL12 is secreted from various tissues, including the tumor, intestine, skin, ovary, bone marrow, liver, lung, kidney, and heart, and has been confirmed to recruit intravenously injected MSCs expressing CXCR4. 70,91,92Recent studies have investigated various methods for enhancing the migration and therapeutic efficacy of MSCs by increasing CXCR4 expression, such as hypoxia, chemical compounds, or cytokines, during MSC expansion in vitro. 93Notably, hypoxic pretreatment has been found to increase CXCR4 expression, promote migration, and reduce the apoptosis of MSCs in vitro. 94In addition to its role in migration and survival, the CXCL12-CXCR4 axis is also critical for MSC cytokine secretion. 95hers.MSCs overexpressing CCR5 exhibit enhanced migration following transplantation into the degenerating retina. 96In mice with allergic contact dermatitis, CXCL10 and CXCL13 mRNA levels were upregulated in inflamed ears.The number of MSCs overexpressing CXCR3 or CXCR5 increased in these ears, indicating that the CXCL10-CXCR3 and CXCL13-CXCR5 axes enhance MSC migration. 97Transwell assays revealed that CXCR6 is involved in the transmigration of MSCs across the EC layer in response to CXCL9. 76Exposure of MSCs to hypoxia increased CX3CR1 expression and migration in vitro. 98ntravenous injections of CX3CR1-overexpressing MSCs led to the generation of long-lasting MSCs in the inflamed colon of mice, even more so than in the liver and lung. 99trix metalloproteinase family Traversing the protein fibers of the ECM, which is present in all tissue types, is crucial for cells to reach target sites in circulation.
Basement membranes separate the epithelium or endothelium from the stroma. 100MSCs break down basement membranes by secreting matrix metalloproteinases (MMPs), which are involved in various physiological and pathological processes because of their capacity to degrade ECM components. 101,102MMPs can also function as motility markers of MSCs, influencing their homing efficiency (Table 3).Gelatinases, MMP-2, and MMP-9 are major contributors to the breakdown of basement membranes. 103owever, MSCs secrete MMP-2 but not MMP-9, identifying MMP-2 as the key player involved in MSC transmigration and invasion. 101Knocking down MMP-2 significantly reduced the transendothelial migration ability of these cells. 104Chemerin, a chemoattractant produced by the liver, adipocytes, and lung, manipulates the transmigration of intravascularly administered MSCs by upregulating MMP-2 expression. 105MT1-MMP, a membrane-tethered metalloenzyme, not only regulates MSC trafficking through the interstitial ECM both in vitro and in vivo but also directs MSC differentiation programs. 106PDGF-BB stimulates MSC migration and proliferation, upregulating MT1-MMP expression on MSCs. 107he expression of four tissue inhibitors of metalloproteinases (TIMPs), which inhibit the active form of all MMPs, is induced by inflammatory cytokines or chemokines. 108Silencing of TIMP-1 enhanced MSC migration, indicating that both TIMP-1 and TIMP-2 play opposing roles in the homing process. 109Additionally, TIMP-1 activity is responsible for the antiangiogenic effects of MSCs in inflamed lymph nodes of mice. 110TIMP-3, produced by MSCs, has been recognized as a soluble factor that positively impacts EC function in a mouse model of traumatic brain injury. 111TIMP-4 has been found to be expressed at very low levels in BM-MSCs. 112rategies to improve MSC homing The success of MSC therapies is facilitated by the effective delivery of living cells to injured tissues to carry out their biological functions.Beyond the exclusive use of anticoagulants, 26 various bioengineering strategies (Table 3), including magnetic guidance, radiotherapeutic techniques, genetic modification, enzymatic modification, protein/antibody conjugation, and priming, have been extensively developed and applied to enhance MSC homing efficiency. 27,38Genetic modification methods, such as viral or mRNA transfection to transiently or permanently overexpress specific molecules governing MSC motility, have been discussed in the previous sections.
Unlike genetic modifications, cell surface engineering aims to chemically alter the surface of MSCs directly through enzymatic modification and protein/antibody conjugation, resulting in temporary but effective enhancements in homing.One such strategy is the enzymatic modification of CD44, which converts CD44 to HCELL through sugar modifications, thereby enabling MSCs to home to the BM via E-selectin and L-selectin. 113This modification also increases MSC infiltration into pancreatic islets threefold following intravenous administration and durably reverses hyperglycemia. 114Attaching anti-ICAM-1 or anti-VCAM-1 antibodies to MSCs has been shown to improve retention and homing to target tissues. 115,116The conjugation of E-selectinbinding peptides onto MSC membranes is utilized to manipulate cell-microenvironment interactions, particularly for targeting cell delivery to specific tissues. 117Gundlach et al. developed a bispecific antibody designed to bind CD90 on MSCs and myosin light chain 1 on ischemic myocardium. 118riming MSCs with small molecules or soluble factors is a simple and effective strategy to boost the molecules that regulate MSC motility.The upregulation of CXCR4 and MMP-9 in MSCs treated with valproic acid and lithium results in enhanced MSC homing, improved functional recovery, and reduced infarct volume in the brains of rats in a cerebral ischemia model. 119In addition, individual MSCs were encapsulated in hydrogels or microgels to increase the residence time of MSCs in target tissues.Mao et al. demonstrated that encapsulating MSCs in alginate-poly-d-lysinealginate microgels significantly improved the delivery efficiency of MSCs to target tissues.Magnetic labeling of MSCs has been explored as a strategy for targeted tissue delivery. 120The enhanced homing ability of MSCs labeled with iron oxide was observed to be ten times greater when these cells were intravenously infused into a rat model.One week post-injection, magnetic MSCs demonstrated improved penetration into both the inner and outer retina in comparison to nonmagnetic MSCs. 121nderstanding the roles of various molecules expressed or secreted by MSCs is crucial for MSC engineering.Bioengineering strategies not only improve the efficacy of MSCs but also accelerate their accumulation in target tissues.These strategies can enhance clinical outcomes by overcoming the challenges of limited MSC persistence and inadequate homing to targeted sites.

TARGET ORGANS OF MSC HOMING AFTER SYSTEMIC ADMINISTRATION
The localization of MSCs within various organs post-administration provides critical insights into their interactions with tissues and target cells, which are pivotal for the efficacy of MSC-based therapies. 122MSCs have the capacity to home to inflammatory or injured sites, and their biodistribution varies due to their diverse pathogenesis in different disease models, where chemokine-chemokine receptor axes play an important role (Fig. 1).

Brain
Although MSCs can cross the blood-brain barrier (BBB), no signal from cells can be detected by any of the tracking modalities in the healthy brain after intravenous injection. 123The biodistribution of MSCs in injured brains is completely different from that in healthy brains.MSCs migrate to injured brains at 4 h and reside in the brain until day 11, which is the endpoint of detection after intravenous administration. 124The homing efficiency and retention time are diverse due to the application of different tracking techniques and administration routes.Oshra et al. monitored the biodistribution of MSCs by a nanoparticle-based computed tomography (CT) imaging technique after orthotopic injection.Gold nanoparticle-labeled MSCs could be detected at 1 h, 24 h, and one month post-injection.However, another study showed that no signal from MSCs can be tracked only on day 2 after intra-Signal Transduction and Targeted Therapy (2024) 9:242 arterial transplantation. 125After using mannitol to enhance the penetrability of the BBB, MSCs could be detected on day 7 and day 28 after infusion compared with those in the control group without mannitol pretreatment. 1268][129] To improve the homing efficiency of MSCs to injured brains, various strategies can be employed for the modification of MSCs prior to administration.The overexpression of CXCR4 or CCR2 in MSCs is used mostly to increase the targeting ability of cells after intracerebroventricular injection and carotid injection. 69,130Shahror et al. demonstrated that MSCs overexpressing growth factor 21 exhibit enhanced homing efficiency in the damaged brain, thereby improving the therapeutic effects on traumatic brain injury. 131Inflammatory cytokines can also improve the homing ability of MSCs.For example, the upregulation of IL-8 in injured brains has been confirmed, which has been shown to improve the homing ability of BM-derived stem cells. 132urthermore, pretreatment of MSCs with other soluble factors and chemical agents can also enhance their ability to home to injured brains.Valproate and lithium have been shown to boost the accumulation of MSCs in the tissue of injured brains. 119Other factors, such as IL-3, IL-6, IL-1β, and IFNγ, can also promote the migration of MSCs to injured tissues. 133In addition to gene modification and chemical pretreatment, physical modification can improve the therapeutic efficacy of MSCs by enhancing the ability of cells to home to the brain.More polycluster superparamagnetic iron oxide nanoparticle-labeled MSCs accumulate in the injured brain after intravenous injection and have longer retention times in the brain than control MSCs. 134lon Numerous studies have shown that MSCs accumulate preferentially in the lungs and do not migrate to the colon after intravenous injection in a normal mouse model. 135However, the homing of MSCs to damaged colons was increased in a dextran sulfate sodium-induced colitis model. 136MSCs were monitored by in vivo optical bioluminescence imaging at 48 h after administration, and imaging revealed an increased number of MSCs in the inflamed colon in colitis mice (1.5-5.5%) in comparison with that in healthy mice (0.3-1%). 137he SDF-1/CXCR4 axis, CXCL2/CXCR2 axis, and CXCL5/CXCR2 axis can play critical roles in the homing of MSCs to the injured colon. 87,138Another study revealed that upregulating CX3CL1 in injured colon tissue and transducing MSCs with lentivirus expressing CX3CR1 markedly enhanced cell accumulation at injured colon sites after 2 h, 24 h, and 8 days intravenous injection. 99Although more MSCs home to damaged colon sites in disease models than in healthy models, the homing efficiency remains insufficient to achieve optimal therapeutic effects.A series of modification strategies could be employed to improve the migration of MSCs to the inflamed colon.Fu et al. constructed dual-functionalized MSCs that overexpress CX3CR1 and IL-25, which can promote the delivery of MSCs to inflamed colons and improve therapeutic effects on inflammatory bowel disease. 99mong the chemokine receptors, CXCR2 plays an important role in regulating the migration of MSCs to damaged tissues, and transient CXCR2 expression on MSCs can enhance the migration of cells to damaged colons. 87When coupled with an anti-VCAM-1 antibody, MSCs had greater migration efficiency than control MSCs and completely attenuated colitis. 139The expression of CXCR4 can be upregulated by pretreating MSCs with IL-1β, which can enhance the migration rate of cells to the colon. 140In addition to IL-1β, IFNγ can also increase the homing of MSCs to injured tissues of the colon. 141The upregulation of ICAM-1 or CXCR4 demonstrated a positive regulatory relationship with the homing efficiency of MSCs to the injured colon. 142Chemical methods for improving the targeting ability of MSCs have been utilized in many studies.Polyribocytidylic acid, the ligand of Toll-like receptor (TLR3), has been confirmed to promote MSC homing by activating TLR3. 143art Intravenously administered MSCs can be detected in infarcted hearts on days 0.5, 1, 2, 4, 8, and 16, peaking on day 1 and then decreasing with time. 144Another study revealed that less than 1% of administered MSCs accumulate in hearts under normal conditions; however, more than 3% of intravenously injected cells could still be monitored in infarcted hearts after 3 months. 145ndrzejewska et al. also demonstrated that MSCs can be tracked in injured heart regions for up to 7 days following intravenous injection. 607][148][149] Overexpression of protein kinase C ε and monocyte chemotactic protein-3 (MCP-3 or CCL7) can improve both the capacity of MSCs to migrate to infarcted hearts and their therapeutic effects. 71,150In addition, coupling MSCs with a palmitated peptide resulted in an increased number of MSCs in ischemic heart tissue compared with noncoated controls. 151dney Only 5.4% of administered MSCs migrated to healthy kidneys after intravenous injection, but treatment with cis-platinum increased the release of chemokines from injured kidneys, enhancing MSC homing to 6.4%. 152Furthermore, the difference in the distribution of MSCs is partly attributed to the route of administration, with more MSCs accumulating in injured kidneys 15 min after intraarterial injection than after intravenous injection in a mouse acute kidney injury model.Most MSCs disappeared within 2 days, with only a barely detectable positive signal remaining in both groups. 153,154Another study reported that more MSCs could be detected in injured kidneys than in normal kidneys 2 h after tail vein injection 155 ; these MSCs exhibited longer retention times and primarily accumulated in the glomeruli of the injured kidneys. 156merging data show that the CCL2/CCR2 axis and SDF-1/CXCR4 axis participate in the migration of MSCs to the kidney. 32,157The therapeutic efficacy of MSCs is closely associated with the number of MSCs migrating to the injured region of the kidney.Many methods for enhancing the homing efficiency of MSCs have been reported.CXCR4 overexpression significantly increased the migratory capacity of MSCs in injured kidneys and has been used in many disease models. 158When coupled with kidney injury molecule-1, MSCs migrate to ischemic kidneys more and reside in kidneys for a longer time. 1591][162] Additionally, ultrasound can enhance the homing capacity of MSCs through the use of various nanoparticles in acute kidney injury. 163,164ver Compared to the normal model, the injured liver model exhibited greater homing efficiency (28.7-35.3%). 165Furthermore, in a normal mouse model, no MSCs were detectable at 5 days after intravenous injection. 122However, MSCs were detected in CCl 4injured livers 24 h post-injection, 166 with the greatest homing observed in the injured liver tissues 1 week after intravenous injection. 165,167Furthermore, compared to intravenous injection, hepatic artery delivery shows greater homing efficiency (20-30%) to injured livers. 168he use of inflammatory cytokines, hypoxia, chemical agents, and genetic modifications could improve the homing of MSCs to injured livers.Cytokines play a critical role in the homing of MSCs to injured livers.The upregulation of cytokines such as stem cell factor-1, hepatocyte growth factor (HGF), MMPs, and chemokines in injured livers accelerates their homing to the liver. 169A previous study revealed that the upregulation of CCL2 in damaged livers and the overexpression of its receptor CCR2 in MSCs can enhance the delivery of MSCs to damaged livers. 68,170Transfecting MSCs with lentivirus overexpressing c-Met can improve the survival rate of rats in an acute liver failure model by enhancing the homing capacity of MSCs. 171Activation of the HGF/c-Met signaling pathway can improve the migration of MSCs to injured sites, so overexpression of HGF is desirable for the targeting of damaged liver sites by MSCs. 172Pretreating MSCs with IFNγ can increase the homing ability of MSCs to damaged liver tissues. 173IL-6 can also improve the homing capacity of MSCs in the fibrotic liver. 174[177] Lung MSCs are initially trapped in the lungs after intravenous injection and then redistributed to other organs with high blood flow, such as the liver, primarily because the structural characteristics of the lung microvasculature cause the majority of MSCs to become sequestered there. 33However, the population of MSCs residing in the lungs diminishes due to increased permeability under injured conditions in the initial stage of injection.][180] The regulation of signaling pathways could similarly enhance the homing capacity to injury sites.MSCs with downregulated Hippo signaling, which can promote the migration of cells, attenuate lung injury in ARDS mice. 181With the knockout of vimentin, fewer MSCs colonize injured lungs. 182After transduction with a lentiviral vector carrying the E-prostanoid 2, the capacity of MSCs to home to injured lung tissues improved significantly. 183retreating MSCs with granulocyte colony-stimulating factor can promote the ability of MSCs to home to injured lungs by upregulating the expression of CXCR4. 184Furthermore, the retention time of injected MSCs in mice could be increased via radiation. 185in MSCs are not detectable in healthy mouse skin after intravenous injection.However, increased numbers of MSCs can be detected in injured skin, and this process is mediated by multiple chemokine axes.Alexeev et al. demonstrated the upregulation of CCL27 in wounded skin tissue and the importance of the CCL27/CCR10 axis in the homing of MSCs to damaged skin. 82Increased expression of CXCR6 on MSCs enhanced the accumulation of cells in wound sites on the skin, which was assisted by the upregulation of the ligand CXCL16 in damaged skin. 75In addition, the ability of the CCL2/CCR2 axis and CCL27/CCR10 axis to enhance the homing efficiency of MSCs has been confirmed in several studies. 67,78,186fter pretreatment with inflammatory cytokines, such as IL-17, and plant-derived components, such as protocatechuic acid, cannabinoids, and icariin, a sufficient number of MSCs can migrate to wounded skin. 187,188Junction adhesion molecule A may accelerate the repair of wounded skin and improve the therapeutic effects of MSCs by increasing their ability to migrate to damaged skin. 189

Other organs
The target organs of MSCs also include the ear and ovary.MSCs can be tracked at 24 h after transplantation in the injured ovary. 190nother study showed that MSCs were located in the interstitium of the ovary at 4 weeks after tail vein injection. 191The SDF-1/ CXCR4 axis participates in the homing of MSCs to the ovary. 92aluronic acid improves the homing ability of MSCs to the sites of injured ears. 192Moreover, the glycoengineering of MSCs significantly enhances their homing to tumors. 193verall, after peripheral intravenous injection, MSCs are initially trapped in the lungs, 33 with a smaller portion passing through to reach organs with rich blood flow, such as the liver, 165 kidney, 152 and brain, 123 and minimal numbers of MSCs migrating to the skin. 82Most in vivo distribution studies have been conducted in mice or rats, and given the significant differences in vascular diameters between these animals and humans, these results cannot be directly translated to humans.MSCs labeled with 111 Inoxine show a gradual decrease in lung retention and an increase in liver and spleen retention over time in humans. 194These findings suggest that MSCs may display unconventional pharmacokinetic characteristics, with the levels of inflammatory cytokines in vivo and molecules regulating MSC motility being crucial parameters for pharmacokinetic evaluation.

THE FATE OF MSC AFTER TRANSPLANTATION
Several preclinical MSC therapy studies have demonstrated that MSCs do not persist in the body long-term and are gradually cleared over time. 27,195There are significant differences in the persistence of MSCs in vivo across various experiments.Some studies indicate that MSCs can survive in the body for only 24-28 h, 33,196 whereas other studies have shown that MSCs are still detectable up to 120 days post-administration. 197 The longterm persistence of MSCs in vivo carries the risk of tumor formation, while short-term persistence may restrict their therapeutic efficacy.Hence, understanding the fate of MSCs is essential for assessing their safety and efficacy.While metabolism and excretion represent the fate of conventional drugs, apoptosis, autophagy, differentiation, ferroptosis, phagocytosis, and senescence are the fates of MSCs (Fig. 2).An increasing understanding of the fate of MSCs after intravenous injection has advanced further research into the mechanisms underlying their therapeutic effects.

Apoptosis
Apoptosis, a programmed cell death process, is mediated by diverse signaling pathways and is triggered by multiple factors, such as cellular stress, DNA damage, and other proapoptotic agents. 198MSCs undergo apoptosis in different target organs after administration, whether in a physiological or pathological environment.He et al. investigated the apoptosis of MSCs after infusion in a mouse liver and spinal cord injury model and attributed the therapeutic effects to the release of phosphatidylserine from apoptotic cells.Apoptotic MSCs were first detected at 2 h post-injection in the liver and lung and markedly decreased at 12 h and 24 h, respectively. 72Another study revealed that in a GvHD model, MSC apoptosis was induced by perforin released from cytotoxic cells one hour after injection, and the MSCs were primarily localized in the lung and spleen.Subsequently, the phagocytosis of apoptotic MSCs by macrophages is essential for the immunosuppressive effects of MSCs, as it leads to the production of indoleamine 2,3-dioxygenase. 8However, Swee et al. reported that MSC apoptosis occurs not only in a pathological environment but also in a physiological state after infusion.After intravenous injection, MSCs rapidly undergo apoptosis in the lungs of recipient mice.Furthermore, the results suggested that apoptosis is not dependent on host cytotoxic or alloreactive cells. 199he apoptosis of MSCs, a complex process influenced by various factors, is driven by inflammatory cytokines such as IFNγ and TNFα, which induce the upregulation of inducible nitric oxide synthase and the production of nitric oxide through the activation of the STAT1 signaling pathway. 200,2013][204][205] The overexpression of certain microRNAs under pathological conditions can initiate apoptosis by downregulating antiapoptotic protein expression, as demonstrated by a study in which miRNA-98-5p triggered apoptosis in MSCs by targeting insulin-like growth factor type 2 mRNA-binding protein 1 and modulating the PI3K/AKT and p53 signaling pathways. 206[209][210][211] Autophagy Autophagy plays an important role in maintaining the therapeutic effects of MSCs. 212As one of the intracellular degradation systems, autophagy is a complex process composed of a variety of signaling pathways that can be triggered by diverse inducers. 213mong the diverse fates of MSCs, autophagy can be activated by various factors, such as cell starvation, inflammation, and oxidative stress. 214MSCs were detected in the lungs at 24 and 48 h postadministration, with a significant increase in the autophagy marker microtubule-associated protein 1 light chain 3 (MAP1LC3).However, MSCs were not detected in the spinal cord until 72 h post-injection, at which time they could still be detected 120 h later, accompanied by a significant increase in MAP1LC3. 215A reduction in IGF-1 expression results in the downregulation of the AKT/mTOR signaling pathway and the upregulation of autophagy in aged MSCs exposed to hypoxic conditions. 216Autophagy also enhances MSC survival in vitro under conditions of starvation, hypoxia, or exposure to ROS. [217][218][219] Differentiation MSCs, a heterogeneous subset of multipotent stromal stem cells, can differentiate into adipocytes, osteocytes, and chondrocytes.Emerging studies have shown that MSCs can also differentiate into ECs and neurons at injury sites after transplantation. 12herefore, the differentiation potential of MSCs is vital for their therapeutic effects.Some MSCs express EC-specific markers after intracerebral administration in a rat model of stroke, revealing that the increase in vascular density is mediated by the differentiation of MSCs into ECs. 220Another study revealed that human adult dental pulp MSCs preferentially differentiated into astrocytes after intracerebral transplantation in a focal cerebral ischemia model. 221nly a few MSCs differentiate into other cells, which mainly occur in the bone, brain, and blood vessels after administration via different pathways. 222he differentiation of MSCs is regulated by various signaling pathways that intricately coordinate this complex process.Intraflagellar transport 20 plays a critical role in determining MSC fate, and its depletion results in a marked increase in adipocyte differentiation. 223In contrast, the suppression of sexdetermining region Y box 9 protein hinders the adipocyte differentiation of MSCs through the inhibition of the transcription factor CCAAT enhancer binding protein β. 224 The interaction between integrator complex subunit 7 and ATP-binding cassette subfamily D member 3, along with the presence of serum IGF-1, facilitates the osteoblast differentiation of MSCs, offering potential for treating osteoporosis. 225,226rroptosis Ferroptosis, which was originally described by Dixon et al. in 2012, represents a distinct form of regulated cell death that is mechanistically different from apoptosis. 227This iron-dependent process is characterized by the accumulation of intracellular iron and increased levels of lipid peroxidation. 228,229Ferroptosis is inhibited by the overexpression of NOP2/Sun RNA methyltransferase 5 or ferritin heavy chain/light-chain in MSCs. 230Conversely, the murine double minute 2-TLR4 axis and the prominin2-BACH1 axis can also participate in ferroptosis in MSCs. 231,232The occurrence of ferroptosis in MSCs after transplantation reduces their efficacy, and intervention strategies are urgently needed to improve their therapeutic effect.Chen et al. demonstrated that increased levels of peroxiredoxin-2 in MSCs can improve therapeutic outcomes in a neurogenic erectile dysfunction rat model by suppressing ferroptosis. 233[236] Phagocytosis MSCs have been widely recognized as promising tools for treating autoimmune diseases because of their ability to cross-talk with various immune cells.Several experiments revealed that nearly half of MSCs were subjected to phagocytosis after intravenous injection. 237MSCs are usually trapped in the lungs and disappear within a day under normal conditions.The fate of MSCs after their disappearance from the lungs was elucidated, showing that MSCs were primarily phagocytized by blood-derived monocytes in the lungs and subsequently redistributed to the liver. 195Another study revealed that MSCs are injured upon contact with blood compounds due to the complement system, 238,239 and this apoptosis and injury facilitate their phagocytosis by monocytes. 8n addition to monocytes, tissue-resident macrophages can also phagocytize intravenously injected MSCs.In a mouse model of asthma, MSCs were primarily localized in the alveolar and capillary walls 1 h post-injection and were phagocytized by lung-resident macrophages 24 h post-injection. 240Furthermore, MSCs can be phagocytized by splenic macrophages at 3 h and 24 h after intravenous injection in tumor-bearing mice. 241nescence MSCs tend to become senescent after injection due to crosstalk with the microenvironment, which hinders their clinical application and leads to undesirable therapeutic effects.MSCs become senescent with increased expression of senescence-associated genes during long-term in vitro culture and due to replicative senescence and their niche after in vivo administration. 242The mechanisms and induction factors of senescence have been described in many studies.ROS not only induce autophagy but also promote the senescence of MSCs. 243,244D-galactose (D-gal) can promote intracellular ROS generation, which markedly induces cell senescence.Coenzyme Q10 and ascorbic acid inhibit the pro-senescence effect of D-gal on MSCs through AKT/mTOR signaling. 244,245In addition to ROS, microRNAs can induce cellular senescence, and inhibiting microRNA-45 and microRNA-155-5p can rejuvenate senescent MSCs. 246,247Overexpression of apelin and Erb-B2 receptor tyrosine kinase 4 could rejuvenate senescent MSCs via the PI3K/AKT and AMPK signaling pathways after administration and enhance the therapeutic efficacy of MSCs in a myocardial infarction model. 248,249Knockdown of CD26 reduces senescence-associated cytokine secretion and potentiates the therapeutic effects of MSCs by delaying the senescence of MSCs in a mouse emphysema model. 250remature senescence of MSCs, which occurs more frequently during the pre-processing and early post-injection stages than natural senescence, remains a significant hurdle in clinical application. 251Multiple signaling pathways are involved in the regulation of the premature senescence of MSCs, with the ROS/mTOR/4EBP1/ p70S6K1/2 pathway and the FoxO3a pathway playing key roles in this process. 251,252Many strategies have been employed in studies to prevent premature senescence as much as possible and improve the therapeutic efficacy of MSCs.Walnut kernel oil and defatted extracts enhance MSC stemness and delay senescence. 253Depletion of GATA binding protein 4 can hinder SASP-dependent senescence in MSCs by suppressing the NF-κB pathway. 254urrently, the apoptosis, autophagy, ferroptosis, and other fates of MSCs have been investigated in vitro.However, many findings on the in vivo fate of MSCs after injection are unknown, which limits the detection and evaluation of MSC efficacy to an extent.In the future, elucidating the fate of MSCs within various target organs in vivo will be crucial, necessitating the development of new probes and biotechnology for such investigations.

THE IMAGING AND TRACKING MODALITIES OF MSC
The homing and persistence of transplanted MSCs in vivo remain poorly understood due to technical limitations, which hinders their clinical translation.Precise and effective detection methods are vital for exploring the migration and distribution of MSCs.Current methods for imaging and tracking in vivo include gene quantitative detection, optical imaging, magnetic resonance imaging (MRI), nuclear medicine imaging, ultrasound imaging, and photoacoustic imaging (Fig. 3), 255 each of which has advantages and disadvantages (Table 4). 256To precisely and effectively detect the biodistribution of MSCs in vivo, a combination of different approaches is required.

Methods for detecting MSC pharmacokinetics
Flow cytometry, immunohistochemistry (IHC), and real-time quantitative polymerase chain reaction (Q-PCR) detection systems are commonly used for the invasive detection of MSCs. 257,258With convenient operation and high sensitivity, Q-PCR can detect and quantify MSCs quickly without labeling, but it cannot distinguish between living and dead cells. 259Flow cytometry is performed by collecting limited tissues for analysis and requires labeling with specific antibodies, which is less accurate and stable than Q-PCR. 260Although IHC can directly detect the distribution of MSCs, it cannot achieve long-term in vivo tracking of cells. 261n vivo tracking of MSCs involves direct and indirect labeling, commonly using radioisotopes, magnetic nanoparticles, and fluorescent dyes for direct labeling.After incubation with the above labeling materials and administration to animals, MSCs can be detected by positron emission tomography (PET)/singlephoton emission computed tomography (SPECT), MRI, and in vivo fluorescence imaging.These tracking technologies can noninvasively and continuously monitor the dynamic distribution of MSCs in preclinical and clinical trials. 256In addition, superparamagnetic iron oxide nanoparticles are used most often in MRI technology, 262 and the radioisotopes 64 Cu-PSTM, 18 F-FDG, 111 Inoxine, and 99 mTc are the primary tracer agents used in PET/SPECT. 263Although the direct labeling method is more convenient, markers are diluted through cell division and shed from cells over time, reducing the accuracy of long-term tracking.
Indirectly labeled MSCs are tracked by detecting fluorescent proteins or specific proteins combined with a nuclide probe, which are transferred into cells by gene modification.Optical imaging technologies, classified as fluorescence imaging and bioluminescence, are popular for tracking living cells.Fluorescence imaging (FLI) detects cells by fluorescence microscopy and mainly monitors the distribution of cells in animals because of limits on detection depth. 264Bioluminescence-based imaging (BLI) is a minimally invasive technique for experimental animals in which luciferase is continuously and stably expressed in MSCs. 265ptical imaging Based on the detection of light emission from molecules after excitation via an internal charged-coupled device camera, optical imaging, which mainly consists of BLI and FLI, plays an important role in researching the biodistribution of MSCs in different disease models. 266With high accessibility and the possibility of long-term tracking, optical imaging is widely used for tracking MSCs.Compared to nuclear imaging modalities, optical imaging has fewer negative effects on cell proliferation, viability, and differentiation.Furthermore, most of the labeling reagents for optical imaging are suitable for longitudinal detection. 267LI relies on various fluorophores that can be excited by specific wavelength light sources, with the emitted light collected by fluorescence signaling components and transformed into images by signal detection and amplification components. 268The exogenous labeling of MSCs using fluorescence has been recognized as an important imaging approach in vivo with excellent accessibility and operability.One of the conventional exogenous fluorescent dyes is hloromethyl-dialkylcarbocyanine, which is frequently used to track labeled cells, especially those that bind to the cell membrane. 269In addition, quantum dots are nanomaterials with intense fluorescence and diverse colors that can directly label MSCs, offering advantages over conventional fluorescent agents. 270To enable long-term cell tracking without affecting cell proliferation and potency, novel fluorescent probes are essential.Liu et al. designed a biocompatible and photostable nanoprobe made of polycaprolactone and di(thiophene-2-yl)diketopyrrolopyrrole that retained strong fluorescence for up to 4 weeks for MSC differentiation tracing. 271However, one drawback of labeling MSCs directly with fluorescent reagents is a false positive signal.When transfected with fluorescent protein plasmids or virus vectors, MSCs have a greater signal-to-noise ratio than when directly labeled with fluorescent agents.The biodistribution of MSCs labeled with green fluorescent protein has been tracked, and the action of these cells post-administration have been characterized. 272,273Compared with other fluorescent reagents, near-infrared (NIR) fluorescent dyes with emission wavelengths ranging from 740 nm to 1700 nm exhibit reduced tissue absorbance, scattering, and autofluorescence, thereby enabling greater depth of penetration.After being labeled with NIR, MSCs can be continuously traced in vivo for up to 21 days, and liver regeneration can be visualized in an acute liver failure model. 274LI relies on the photon emission generated by the catalytic conversion of luciferase, which facilitates the intramolecular oxidation of luciferin in the presence of ATP and oxygen within luciferase-expressing MSCs. 2757][278] BLI can also avoid interference from background and biological autofluorescence signals during the imaging process.
In summary, optical imaging is widely used for MSC tracking due to its long-term tracking ability, lower cytotoxicity compared to nuclear imaging, diverse fluorescent reagents, and accessibility.However, its poor tissue penetrability and light scattering limit its use in small animal imaging (Table 5).

Nuclear imaging
Nuclear imaging tracks the biodistribution of MSCs by imaging tracers that have been labeled with radionuclides.There are three nuclear imaging techniques: PET, SPECT, and planar gamma scintigraphy. 279PET and SPECT will be summarized in this review since they are used more widely than planar gamma scintigraphy in the tracking of MSCs.
The gamma-ray photons with well-defined energy levels emitted from SPECT radioactive tracers can be captured directly by a gamma camera, where they are eventually digitized to provide 3D information on the biodistribution of MSCs 279 Although the SPECT imaging modality offers relatively high sensitivity and quantifiability, its widespread application in the long-term tracking of MSCs is limited due to the use of radiotracers with short half-lives.99m Tc, which has a half-life of only 6 h, can be detected persistently for 3 days in vivo after injection. 280Compared with 99m Tc, 111 In-labeled MSCs can be detected up to 14 days after intravenous injection because of their long half-life (t 1/2 = 2.81 days).Another drawback of SPECT is its cytotoxicity associated with the use of radiotracers.Although it has no impact on the viability or differentiation of human MSCs labeled with 111 In, many other experiments have shown that radiotracer labeling can affect MSC viability and differentiation in a dose-dependent manner. 281Owing to the imaging properties of SPECT, the signal of MSCs in deep tissues could also be captured by a detection camera.When human BM-MSCs were labeled with 99m Tc, their biodistribution in arthrosis was fully depicted using a gamma camera. 282ET imaging involves capturing the signals of gamma photons emitted in opposite directions by positron-emitting radionuclides using a ring detector, followed by data processing and image reconstruction.In contrast to SPECT, the PET imaging modality has higher spatial and temporal resolution but shorter cell tracking time in vivo.However, similar to SPECT, PET imaging is limited by timedependent ionizing radiation injury to target cells and the short halflife of tracers, which restricts its use in long-term cell tracking. 279he isotopic tracers commonly used in PET imaging are 18 F-fluorodeoxyglucose (FDG) and 18 F-FHBG. 270,283Nose et al. tracked the biodistribution of MSCs in multiple animal species using PET imaging with 18 F-FDG following various routes of administration. 284While 89 Zr, which has a longer half-life (t 1/2 = 78.4h) than 18 F (t 1/2 = 109.7 min), can also be used for tracking MSCs, Patrick et al. labeled MSCs with 89 Zr to show its delivery to the lungs in a mouse lung cancer model up to 7 days post-injection. 285While nuclear imaging offers good tissue penetrability and sensitivity, its limited spatial resolution and short imaging time make it unsuitable for precise and longitudinal MSC imaging (Table 5).
Magnetic resonance imaging MRI is a powerful imaging tool with superior spatial resolution and tissue penetrability under preclinical and clinical conditions. 286his modality can also provide 3D information on MSC biodistribution as well as nuclear imaging.8][289] While ferumoxide-labeled MSCs were observed only in the lung, perfluorocarbon-labeled MSCs dispersed to other distant organs. 290Most cell tracking studies use dextran or carboxy dextran-coated SPIONs, which are designed for uptake by phagocytic cells 291 ; however, their impact on MSC proliferation and function must be evaluated.Novel SPIONs coated with aminopolyvinyl alcohol, which offer greater colloidal stability, greater solubility, greater biocompatibility, and lower toxicity, have been developed and are primarily taken up by MSCs. 292espite its high spatial resolution and tissue penetrability, MRI has low sensitivity and long imaging times, making it difficult to track labeled cells throughout the whole body and determine their migration beyond damaged tissue.Furthermore, the accessibility of MRI is undesirable due to the high cost of imaging instruments (Table 5).Magnetic particle imaging Magnetic particle imaging (MPI), which usually involves the use of noncytotoxic SPIONs, is a relatively new tomographic imaging technique (Table 5).MPI can detect nanoparticle tracers at any time and space in the body with greater spatial and temporal resolution than MRI. 293Sehl et al. compared the sensitivity of MPI and MRI using perfluoropolyether-labeled MSCs and revealed that MPI reliably detected 4000 MSCs, while MRI detected 256,000 MSCs at the same time. 294Zheng et al. utilized MPI to dynamically track and quantify the biodistribution of SPION-labeled MSCs following intravenous injection in mice.One hour post-injection, MSCs primarily accumulate in the lungs before gradually migrating to the liver within 1 day. 295otoacoustic imaging and ultrasound imaging As a non-invasive and emerging imaging modality, photoacoustic imaging (PAI) offers rich optical contrast, high ultrasonic resolution, and fast data acquisition, making it suitable for real-time imaging (Table 5).Given these advantages, Li et al. demonstrated that Prussian blue particle-labeled MSCs were able to cross the BBB and subsequently migrate to injured brain regions. 296 5).Since a single imaging modality cannot simultaneously meet all these requirements, multiple-modality imaging has emerged in recent years. 33ombining nuclear imaging with bioluminescence-based imaging enables long-term cell tracking and is complemented by anatomical information from CT, effectively offsetting the limitations of each method. 298Ultrasound imaging and PAI offer high contrast, flexibility, and fast acquisition for real-time MSC transplantation guidance, while MRI with anatomical imaging resolution and SPECT with high sensitivity and quantitative capability are ideal for long-term monitoring of cell biodistribution and migration in vivo.Yao et al. combined these modalities to monitor engrafted MSCs in real-time and track their long-term biodistribution. 299The optical-MRI modality can improve the sensitivity of MSC detection in vivo and the resolution of imaging. 255n recent years, in vivo tracking imaging technologies have developed rapidly.Compared with single detection technology, employing multiple methods provides more comprehensive monitoring of MSC distribution in vivo.By combining Q-PCR with various imaging techniques, the dynamic spatial distribution of living cells, along with anatomical structures and molecular phenotypes at single-cell resolution, can be obtained.This approach provides robust data to support the evaluation of the efficacy of MSC therapies.Furthermore, with the advancement of multimodality imaging techniques, it is essential to develop nanoprobes with multiple imaging properties to enable effective imaging of MSCs after transplantation.This is crucial for the clinical tracking and application of MSCs.

DRUGGABILITY RESEARCH FOR CLINICAL TRANSLATION
Like conventional drugs, MSCs also adhere to the guidelines of safety, effectiveness, and quality controllability.Despite their remarkable efficacy in preclinical animal trials, MSC therapies have faced challenges in clinical translation due to individual variability and the considerable number of non-responsive patients. 300everal factors may lead to suboptimal clinical outcomes, including heterogeneity in MSC product potency, pharmacokinetics across different administration routes and a limited understanding of how host responses post-administration impact therapeutic efficacy. 301lucidating the relationships among the disease state, biological distribution, and efficacy of MSCs in vivo helps address a significant challenge to their clinical application.Preclinical research on the druggability of MSCs should not only clarify the relationship between their dynamic biodistribution and efficacy but also elucidate their mechanisms of action and develop reliable biomarkers.These efforts will improve patient stratification and facilitate the establishment of precise therapeutic strategies, ultimately optimizing clinical efficacy (Fig. 4).

Guidelines and consensus
Conventional preclinical and clinical evaluation methods are inadequate for cell-based products due to the differences between cells and conventional pharmaceuticals.The use of MSCs and other cell therapies is under the regulatory oversight of the Center for Biologics Evaluation and Research, specifically the Office of Cellular, Tissue, and Gene Therapies in the United States.MSCs and other cell therapies are categorized under Advanced Therapy Medicinal Products in the European Union.Japan regulates medical products derived from human cells, genes, or tissues under the Pharmaceuticals and Medical Devices Act (PMD Act). 302In China, all cell therapies, including MSC therapy products, immune cell products, and gene-modified cell products, can be found in the Technical Guidelines for Research and Evaluation of Cell Therapy Products issued by the NMPA in 2017.The NMPA updated the Biological Products Appendix of the GMP for Drugs in 2020 and drafted the Appendix for Cell Therapy Products in the draft version of the GMP for Drugs.Furthermore, the NMPA drafted the Technical Guidance Principles for Clinical Trials of Human Stem Cells and Their Derived Cell Therapy Products on August 4, 2020.New drug development must adhere to the current Chinese Pharmacopoeia and other national drug standards. 303[305] Quality control of MSC therapies Although the translation of MSCs from bench to bedside is feasible, most MSC therapies face unsuccessful late-stage clinical trials, with notable safety profiles but often limited efficacy in humans.These failures emphasize the challenges associated with the quality control of MSCs for clinical trials. 18Given the high heterogeneity and complex biology of MSCs in vivo, enhancing manufacturing processes and formulation technologies and establishing standardized production systems and personalized quality control are essential for successful clinical trials. 301he International Society for Cell and Gene Therapy (ISCT) initially established three minimum criteria for human MSCs based on their morphology, surface markers, and trilineage differentiation.306 MSCs are defined as adherent cells with a spindle-shaped morphology under standard culture conditions.These cells exhibited the following characteristics: (1) expression of CD105, CD73, and CD90 but lacking expression of CD45, CD34, CD14, or CD11b, CD79a, CD19, or HLA-DR; and (2) the capacity for differentiation into osteoblasts, adipocytes, and chondrocytes in vitro.307 These criteria reflected the "stemness" of MSCs rather than determining their therapeutic properties.Given the variations that exist at multiple levels, these criteria were insufficient to define MSCs comprehensively.In 2019, ISCT updated its standards for defining MSCs to include tissue origin and relevant functional assays to clarify their associated therapeutic mode.Furthermore, regulatory agencies mandate the identification, viability, purity, potency, proliferative capacity, genomic stability, and microbiological testing of MSCs.308,309 The aforementioned criteria do not encompass the efficacy of MSC therapies; quality control of MSC therapies also includes the following: (1) the source of MSCs, considering the health status, genetics, gender, and age of donors, as well as diverse tissue origins 1,310 ; (2) the methods employed for isolating and obtaining cells from these tissues, whether through enzymatic or mechanical dissociation, which can impact the efficacy of MSCs 311 ; (3) culture conditions, including aspects such as the composition of the culture medium, oxygen levels, flask/bioreactor, passage number, and cell surface modifications, which may similarly affect the efficacy and homing capacity of MSCs 308,312 ; (4) the techniques used for cryopreservation and subsequent thawing/culture rescue, which can influence the viability, function, and homing potential of MSCs [313][314][315] ; and (5) the expression of biomarkers for efficacy and target tissue homing, such as paracrine effectors and chemokine receptors.316,317 Pharmacokinetics of different sources The biodistribution of MSCs can be influenced by various factors, such as cell type, route of administration, and host immune status. MSC from different tissues are capable of trilineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) and exhibit similar surface markers, but significant differences still exist in terms of their biological characteristics and functions.318 MSCs isolated from BM and adipose tissues express the same surface biomarkers but differ in the expression of cell adhesion molecules such as integrin α4, ICAM-1, CD34, and VCAM-1.319 Li et al. compared the proteomes of MSCs derived from the BM, placenta, and umbilical cord to investigate their expression of migrationrelated proteins, and the results showed that the migration ability of UC-MSCs was weaker than that of MSCs from the BM and placenta (5.9-fold and 3.2-fold, respectively).320 However, Hori et al. reported that UC-MSCs exhibit greater migration toward lymphocytes than do BM and adipose tissue-derived MSCs.321 These differences may be attributed to variations in chemokine receptors among MSCs from distinct tissue sources.
The differences between autologous and allogeneic MSCs extend to their pharmacokinetics.The lower immunogenicity of autologous MSCs enables them to rapidly localize to target tissues or organs without encountering immune rejection.Consequently, they exhibit prolonged survival in the body due to reduced susceptibility to host immune attack and clearance. 322,323However, the application of allogeneic MSCs is encouraged because of factors such as their source and quantity.The industry-sponsored production of allogeneic MSCs enables the manufacture of up to 1 million doses per donor for widespread deployment, far exceeding the capacity of autologous MSCs, 324 indicating that this choice is not solely driven by biological advantages.

Preclinical pharmacokinetic research
Pharmacokinetic studies of MSC therapy, which track the biodistribution and fate of cells, play an indispensable role in elucidating the in vivo processes and associated biological actions, as well as in explaining the safety and efficacy of cell therapy products.The pharmacokinetic studies also evaluated the dose concentration, interval and route of administration.The Technical Guidelines for Research and Evaluation of Cell Therapy Products (China) emphasize the application of one or more methods to monitor the dynamic distribution, migration, homing, survival, and extinction of cell therapy products.Additionally, preclinical pharmacokinetic research should focus on the physiological processes of MSC therapy products in vivo, examining their differentiation capabilities and the biological effects related to their biodistribution. 303,305Although none of the agencies recommend methods for analyzing the spatial and temporal distribution of cells, a fundamental principle is that the methods Examples of such methods listed in the guidelines include MRI, PET, SPECT, FLI, autoradiography, PCR, IHC, and in situ hybridization. 264Moreover, the selection of appropriate animal models should align with the pathogenesis and therapeutic mechanism of MSCs. 264In addition to examining the conventional distribution and migration of MSCs, it is important to consider the impact of gene modification or bioengineering approaches on MSCs, their expression or secretion of biomolecules, and their interactions with host cells, which involve the tissue responses induced by the secretion of bioactive molecules from MSCs. 304The preclinical pharmacokinetic characteristics of MSCs can help inform experimental design and patient stratification in clinical pharmacokinetic research.

Clinical pharmacokinetic research
Clinical pharmacokinetic research cannot employ invasive detection methods such as those used in animal experiments.Extrapolating the results of animal studies of MSC therapies to humans is challenging.The mismatch between mouse and human clinical outcomes may arise from differences in immune compatibility, dosage, condition of culture-adapted MSCs, and variations in disease states of the hosts. 18In addition to the aforementioned factors in preclinical pharmacokinetic research, individual variability presents a significant challenge in clinical pharmacokinetic research.The diverse pathological conditions, such as varying levels of inflammation and oxygen, present in clinical subjects greatly impact the pharmacokinetic characteristics of MSCs in the body, thereby influencing the quantity and viability of MSCs that reach specific target organs or tissues. 256Moreover, the interactions between MSCs and host cells lead to variations in their therapeutic efficacy. 325Alsasem et al. developed an advanced, real-time framework for managing MSC transfusions in COVID-19 patients by automatically categorizing patients into various emergency levels and prioritizing them based on their individual status within each level. 326Another important consideration is that the biodistribution of administered MSCs may be influenced by the age of the recipient, which is closely linked to varying metabolic disorders and immunity. 327The pharmacokinetic biomarkers of MSCs identified in preclinical studies can be used to predict and validate clinical pharmacokinetics in patients.The development of therapeutic strategies for individual patients to achieve precise and personalized therapy is emerging as a crucial direction for the future advancement of MSC therapy.

Pharmacokinetics model for systemic administration
To satisfy the clinical requirement for the accuracy of MSC dosage, several pharmacokinetic models have been designed.Previous studies have used a two-compartment pharmacokinetic model, referring to MSCs as inactive and micron-sized nanoparticles, to predict their distribution, yielding remarkably similar data to the actual bioavailability of MSCs post-transplantation. 328 However, this model was unable to predict the precise biodistribution of MSCs due to its disregard of MSCs as a dynamic "living therapy".By integrating anatomical structure into the design, the physiologically based pharmacokinetic (PBPK) model offers greater physiological similarity and prediction accuracy.The first PBPK model of MSCs, reported in 2016, used in vivo optical imaging to track MSC biodistribution and flow cytometry to quantify MSCs. 329himeric antigen receptor (CAR)-T cells are recognized as effective therapeutic agents for antitumour applications.Given the similarities between CAR-T cells and MSCs in terms of therapeutic mechanism and cell properties, the model structure and systemspecific parameters of the CAR-T-cell PK model can be applied to MSCs.PBPK models of CAR-T cells have been developed that incorporate various parameters, such as tissue volume, blood flow velocity, affinity of CAR-T cells for target cells, antigen abundance, and tumor volume. 330To accurately predict the biodistribution of CAR-T cells in vivo, the types of target cells and the expression of ligands on target cells in different disease stages, which could represent the specific properties of CAR-T cells, should be considered in the development and optimization of models. 331he target organ volume, microenvironment, and interaction between MSCs and other cells can all be considered important parameters or processes for PK models.Furthermore, the physical status of donors, tissue resources, passage number of MSCs, administration route, and dose of MSCs can all be recognized as critical covariates in models because of their unignorable influence on the biodistribution of MSCs. 332To maximize the therapeutic effects and minimize the adverse effects of MSCs, a PK model that has a superior ability to predict the biodistribution and number of MSCs in target organs is urgently needed.

CONCLUSIONS
MSC therapy, which has undergone significant progress over the past half-century, has emerged as an effective treatment for major chronic diseases and severe trauma repair in the field of regenerative medicine. 27Although the safety and therapeutic efficacy of MSC therapy have been extensively validated in numerous clinical trials worldwide, only a limited number of MSC products are commercially available in specific regions.This is largely attributed to the challenges associated with pharmacokinetics and the lack of clarity regarding MSC mechanisms, as well as the substantial individualized differences observed in clinical trials.In this review, we discuss MSC homing processes to different organs in vivo and the key regulatory factors involved.We also analyzed MSC fate and evaluated various in vivo tracing methods for detecting MSC homing, highlighting their advantages and disadvantages.Furthermore, we have emphasized the close relationship between the pharmacokinetics of MSCs and their druggability.We have summarized current strategies and suggestions for enhancing the homing capabilities of transplanted MSCs.
MSC therapy products are not subject to conventional pharmacokinetic testing, which is primarily used for conventional drugs.The mode of action of MSCs operates through a "shotgun model" and "hit-and-run" (or "touch-and-go") mechanism: (1) They exert regulatory effects on the body through multiple targets and mechanisms to maintain homeostasis within niches rather than through single-target and simple inhibitory actions 9 ; (2) Many studies have shown their rapid migration to damaged tissue, where they are quickly cleared after stress-induced release of therapeutic molecules, but their therapeutic effects persist over a long period. 12However, there are still many unanswered questions.First, given the high heterogeneity of MSCs, future research should focus on homing studies for different indications to further standardize quality criteria and production processes, ensuring the consistency of MSC product batches.Second, most studies have not linked the fate of MSCs to persistence in vivo due to limitations in detection and labeling technologies.Combining multiple labeling methods and various imaging techniques will facilitate high-resolution imaging and quantitative spatiotemporal analysis of MSCs in target organs at the single-cell resolution level.Third, it is important to note that preclinical mouse models often do not fully replicate human diseases.Although MSCs exhibit very low immunogenicity, they can trigger varying levels of immune activation in both humans and animals.Additionally, the dosage of MSCs administered to mouse models differs significantly from that administered to clinical patients, which can affect treatment outcomes.Finally, screening response patients based on biomarkers to determine the biodistribution and biological mechanisms of MSCs before clinical application can enhance the efficacy of MSC therapies.On the other hand, combining MSCs with gene modification, click chemistry, and tissue engineering materials hold promise for enhancing the precise biodistribution of MSC therapies.This innovative approach has the potential to accelerate the clinical translation of MSCs and expand their application to new therapeutic frontiers, ultimately benefiting patients with a wide range of medical conditions.

2 NCT02491658
Pharmacokinetic characteristics of mesenchymal stem cells in. . .Shan et al. motility, can generate actin-based forces to pull the cell body forward through more stable interactions between MSCs and ECs.

Fig. 1
Fig. 1 Multiple steps in mesenchymal stem cells (MSCs) homing.The homing processes of MSCs are dependent on a series of molecules of motile potency, including selectins, integrins, chemokine receptors, and proteases.Selectins, which are expressed on endothelial cells (ECs), capture MSCs by interacting with their ligands and then mediating the rolling of MSCs.MSCs can thus be activated by exposure to chemokines, consequently leading to integrin-dependent firm adhesion to the endothelium.At the appropriate location, MSCs cross the endothelial barrier by secreting different proteases before continuing chemokine-mediated migration

Fig. 2
Fig. 2 Regulators and effects of different MSC fates.Regulated by a variety of intrinsic or extrinsic factors, MSCs develop into different destinies.MSCs have stem cell properties and diverse differentiation directions under distinct conditions.Phagocytosis of MSCs is mediated mostly by the monocyte-macrophage system.Autophagy and senescence can be activated in ROS-enriched environments or under other conditions.Many MSCs undergo apoptosis.Changes in the functions and phenotypes of MSCs and their consequent effects can be detected when MSCs progress to different fates

Fig. 3
Fig. 3 Multimodal tracing strategies for MSCs in vivo.Precise and effective detection methods are vital for exploring the migration and distribution of mesenchymal stem cells (MSCs).Currently, there are various detection techniques available.Nuclear imaging tracks the biodistribution of cells by imaging radiolabeled tracers.Optical imaging, including bioluminescence imaging (BLI) and fluorescence imaging (FLI), plays an important role in researching the biodistribution of MSCs in different disease models.Cell tracking with magnetic resonance imaging (MRI) requires labeling cells with contrast agents.Magnetic particle imaging (MPI) can detect nanoparticle tracers at any time and location in the body with greater spatial and temporal resolution than MRI.Ultrasound imaging and photoacoustic imaging are noninvasive and emerging imaging modalities.The development of multiple-modality imaging is an emerging trend in MSC tracking and clinical research

Table 4 .
Comparison of technologies for detecting the distribution of MSCs 256 Distinguish live/dead cells; Single-cell resolution Invasiveness; Limit on detection depth, High sensitivity; High specificity; Single-cell resolution Limited detection area; Selective imaging of specific protein; No dynamic detection Pharmacokinetic characteristics of mesenchymal stem cells in. . .Shan et al.

Fig. 4
Fig. 4 Druggability of MSCs.Applying the right treatment to the appropriate population, in the correct dosage regimen, and at the appropriate time is the most crucial principle in the druggability process of MSCs.The biodistribution and fate of MSCs at different time points were monitored by various imaging technologies.These parameters were combined with pharmacodynamic data to construct a PK/PD model of MSCs.Prediction of the pharmacokinetic properties of MSCs in patients, appropriate dosage regimens, and individualized treatments can be achieved with the PK/PD model.Therefore, the most crucial step in the druggability of MSCs is constructing an appropriate treatment regimen based on the pharmacokinetic and pharmacodynamic properties of MSCs

Table 3 .
Molecules governing MSC motility during homing

Table 3 .
continued Pharmacokinetic characteristics of mesenchymal stem cells in. . .Shan et al.

Table 5 .
Methods for tracking MSCs